Avian Influenza, also called “AI,” is an acute and highly contagious viral infection of chickens and other fowl. As an influenza virus, it is classified in subtypes on the basis of antigen differences in the haemoagglutinin (HA; also may be abbreviated as H) and neuroaminidase (NA; also may be abbreviated as N) molecules, which “reassort” or “mutate” from season to season. Because it constantly mutates, vaccine preparation is difficult due to the unpredictability as to which strain will reappear in subsequent seasons. The strains used for vaccine preparation often do not reproduce under manufacturing conditions at a very fast rate, so that waiting for an appearance of a particular strain, and then manufacturing the correct vaccine to protect against the strain does not provide a viable option. Typically, the epidemic of the particular strain will last for several months, and then perhaps disappear for several years.
Eradication is the principal method for controlling the disease in avians, without obvious economic disadvantages, but if a vaccine with a fast onset of immunity could be produced, such a product would offer a viable alternative to mass slaughter of entire flocks.
The influenza viruses are known to be classifiable in the various A, B. C topologies, according to the group antigen the viruses carry. The influenza viruses of the A, B. C types can be distinguished from one another on the basis of the antigen differences that can be found in the viral nucleocapsid (NP) and matrix (M) proteins. In particular, the A-type influenza viruses can be classified into subtypes on the basis of antigen differences in the haemoagglutinin (HA) and neuroaminidase (NA) molecules. Presently nine subtypes of the neuroaminidase NA proteins, designated NA1 to NA 9, and fifteen different subtypes of the serum haemoagglutinin HA proteins, designated HA 1 to HA 15, have been identified. In birds, viruses carrying any of the various HA (or H) and NA (or N) subtypes have been isolated.
HA is a viral surface glycoprotein comprising approximately 560 amino acids and representing 25 of the total virus protein. It is chiefly responsible of adhesion of the viral particle to the host cell and of 25 its penetration into the latter in the early stages of the infection. Haemoagglutinin, among the viral proteins, is the one that is most subject to post-translational rearrangements. After the synthesis thereof has been completed, the molecule follows the 5 exocytotic pathway of the host cell, in the course of which HA is folded, assembled in trimers and glycosylated. Finally it is cleaved into two subunits H1 and H2; this cleavage is the key step in the activation of the molecule and in the acquisition of 10 the infective capacity by the virion.
It is well known, in fact, that the different composition of the cleavage site, concerning the basic amino acid residues, translates into the capacity of the avian influenza virus to produce localized, or symptomatic infections, or, vice versa, generalized infections having a lethal outcome for many avian species. It has therefore been suggested that this fact might influenza the organ-tropism, the host specificity, as well as the pathogenicity of the virus. With respect to the pathogenicity of the virus, strains with multibase-site HA find proteases that cleave the HO molecule, in the active form Hi and H2 in several cellular types thus giving rise to multiple infection cycles with a massive production of 25 infectious viral particles and causing a generalization of the infections in all of the districts within a short time period (HPAI strains). The infection will consequently turn out to have an acute-hyperacute course, with very high mortality.
Neuroaminidase (NA) represents the second membrane glycoprotein of the influenza A viruses. it is coded for by a gene (segment 6 of the virus RNA) of 1413 nucleotide length that codes for a 413 amino acid peptide. This protein has at least two important 10 functions: destruction of the cellular receptor for the viral haemoagglutinin by cleaving between the sialic acid molecule and the haemoagglutinin itself. In this way it is supposed to possibly ease the liberation of the viral progeny by preventing the newly formed viral particles from accumulating along the cell membrane, as well as promoting the transportation of the virus through the mucus that is present on the mucosal surface. NA moreover represents an important antigen determinant that is subject to antigenic variations.
There currently exists a need in the art for avian influenza vaccines which would provide a useful alternative to eradication of infected flocks. Such vaccines would need to elicit a quick immune response in the vaccinated avian, and preferably would enable vaccinated birds to be able to be differentiated from infected birds. Bivalent or polyvalent influenza vaccines have been postulated to be utilizable in the so-called “DIVA” methods, where a vaccine is administered having an N different from the strain being vaccinated against so as to provide a means of differentiating vaccinated from infected birds. Published PCT WO 03/086453, whose disclosure is incorporated in its entirety by reference, describes the DIVA technology, and some representative vaccines utilizable in the methods thereof.
Combined or multivalent vaccines offer a number of obvious advantages over monovalent vaccines. One advantage of a multivalent vaccine is that fewer vaccine inoculations are required. A single preparation can be administered in one inoculation and is effective against several diseases or strains of a single disease. As the range of potential viral strains increases, the combination of vaccines becomes even more mandatory in order to minimize the number of inoculations. The decreased number of inoculations needed when vaccines are combined would likely lead to an increased compliance to the vaccination schedule. This in turn would likely lead to a resulting increase in vaccine coverage, which would ultimately lead to better disease control.
An unexpected problem of combined vaccines is the recently identified negative influence that one vaccine may have on another in a combination vaccine, the so-called “antigen interference” effect. It has been found that when two existing vaccines are simply mixed, one or both may lose their potency (Andre, F. E., “Development of Combined Vaccines: Manufacturers' Viewpoint,” Biologicals 22:317-321 (1994); Hadler, S. C., “Cost benefit of combining antigens,” Biologicals 22:415-418 (1994); Goldenthal. K, L., et al., “Overview—Combination Vaccines and Simultaneous Administration. Past, Present, and Future.” In: Combined Vaccines and Simultaneous Administration, Current Issues and Perspectives (Eds. Williams, J. C., et al.) The New York Academy of Sciences, New York, pp. 1 XI-XV (1995); Clemens, J., et al., “Interactions between PRP-T Vaccine against Hemophilus influenzae Type b and Conventional Infant Vaccines Lessons for Future Studies of Simultaneous Immunization and Combined Vaccines.” In: Combined Vaccines and Simultaneous Administration. Current Issues and Perspectives (Eds. Williams, J. C., et al.) The New York Academy of Sciences, New York, pp. 255-266 (1995); Insel, R. A., “Potential Alterations in Immunogenicity by Combining or Simultaneously Administering Vaccine Component,”. In: Combined Vaccines and Simultaneous Administration. Current Issues and Perspectives (Eds. Williams. J. C., et al.) The New York Academy of Sciences, New York, pp. 35-47 (1995)).
Unfortunately, it cannot always be predicted by the use of currently established potency tests in the laboratory whether individual vaccine components will retain their potency. For example, several independent studies reported that when the Hib vaccine is combined with a whole cell pertussis vaccine there is no interference between the two vaccines but when the Hib vaccine is combined with acellular pertussis vaccines there is a substantial loss of the Hib immunogenicity. It was shown that when Hib is combined with DTaP, it maintains its immunogenicity if given at separate sites, while the immunogenicity is 5-15 times lower when the vaccines administered combined at the same site. This unexpected result confirms that combining two existing vaccines may not be a simple or routine process, and such combination often gives very unpredictable results that are not detected during the initial studies. The studies required to document non-interference often adds several additional months or years of studies to document non-interference, and suitability for use.
Bivalent avian influenza vaccines have been available in the marketplace, such as the vaccine known as Fluvac® marketed by Merial, but there still exists a need for improved bivalent or polyvalent avian influenza vaccines which invoke a rapid immune response, and a higher titre response, and which can thus be utilized to quickly protect birds in the face of an outbreak.